A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

The overall goal of this method is to rapidly and reliably guide muscle fiber quantification in whole rat muscles in an automatic and user-independent manner using an open-source platform. This method can help answer key questions in the neuromuscular field about defects of aging, disease, or trauma on skeletal muscles. The main advantage of this technique is that it can be used to reliably analyze muscle fiber populations and to generate high-quality images of muscle cross-sections.

After sufficient thawing, carefully rinse the specimens in PBS supplemented with Triton X, or PBST, for one 10-and one five-minute wash. After the second wash, air-dry the sections for two minutes. Then, use a hydrophobic pen to encircle the individual cross-sections.

After another 15 minutes of air-drying, block any nonspecific binding with PBST supplemented with goat serum for one hour at room temperature. Next, wash the slides and add the appropriate primary antibody cocktail to each sample for a one-hour staining at room temperature protected from light. At the end of the incubation, rinse the slides with one 10-and one five-minute PBST wash.

Then, label the muscle sections with the appropriate secondary antibody cocktail for one hour protected from light. After washing the slides two times in PBST, as demonstrated, dry the slides off and then stain the nuclei with a DAPI nuclear staining kit according to the manufacturer's instructions. Remove the excess DAPI with a one-minute PBST wash, followed by their brief air-drying.

Then, cover the samples with fluorescent mounting medium and cover slips, and store the slides at four degrees Celsius protected from light. Within 24 to 48 hours of staining, load the slides into a slide scanner and open a new project in the program software. For the slide preview, select the DAPI channel and the 2.5x lens, and manually focus on the first specimen.

Outline each cross-section that should be included for the detailed acquisition process and set the exposure times for each channel. Now, run the automatic image acquisition, checking for the correct acquisition of the first field of view, to prevent an incorrect acquisition early in the automated process. When the image acquisition is complete, manually check that the autofocus of the cross-sections is correct and that the individual fibers are clearly distinguishable.

It is critical that the autofocus is checked and corrected manually for every image as necessary. The automated muscle fiber quantification process requires high-quality images to obtain optimal results. Then, for every cross-section that should be included in the final analyses, export each channel except DAPI separately as JPEG files, and name and sort the files into the appropriately-named folders according to the exposed channels.

Before beginning the muscle fiber quantification process, preview each image in the appropriate image-editing software to confirm a sufficient contrast between the stained fibers and the background. Next, in ImageJ, use the Plugins>Macros>Edit command to reveal the macro source code, and change the folder directory for each channel in the source code of the macro. Now, use the Run command to start the macro.

All of the images in the folder will be loaded into the macro and quantified in a consecutive order. At the end of the quantification, export the results into the appropriate spreadsheet software, and identify the values for the positively-counted slow, intermediate, and fast fibers. The total fiber number can then be calculated.

The immunofluorescent staining of muscle fibers is quick with minor cross-reactivity, and the resulting images demonstrate a high contrast between the stained fibers and the surrounding tissue, as well as a strong distinction between the different fiber types. Automatic analysis allows the detection of the relative muscle fiber populations with an accuracy of plus or minus 4%compared to the manual analysis, with the overall absolute counts higher compared to the manual analysis. The relative counts, however, remain similar, within a plus or minus 4%range.

After watching this video, you should have a good understanding on how to use this method to automize muscle fiber quantification. Don't forget that working with DAPI can be hazardous. Therefore, wear the appropriate protective gear while performing the staining.